This proposal presents a new strategy for the molecular isolation and analysis of megabase regions of human and other genomes. It should also obviate the problem of noncontiguous genomic regions present in many reduced chromosome hybrids. Implementation of this technology should not only facilitate the generation and closure of a map for chromosome 11, but it could be applied to other chromosomes as well. This technique is based upon our finding that gene amplification in mammalian cells is most frequently initiated by a deletion of the corresponding locus from the chromosome. The deletion generates an autonomously replicating extrachromosomal circular molecule. We will use homologous recombination to target a vector containing a dominant acting dihydrofolate reductase (DHFR*) gene into predetermined regions of chromosome 11. Selection with methotrexate will generate cells with amplification of the targeted vector and flanking DNA. The insertion vector also contains one arm of a yeast artificial chromosome (YAC) in order to allow direct rescue of the amplified region without resorting to the construction and screening of an entire library. The presence of amplified copies of the target region will greatly facilitate the rescue from a minimum number of cells. The rapid rescue and mapping features of this strategy should enable us to obtain and characterize the 75-150 homologous integrants which will be required to construct an overlapping map of the 150mb chromosome 11.